Until quite recently, the consideration of oligonucleotides in any capacity other than strictly informational was unheard of. Despite the fact that certain oligonucleotides were known to have interesting structural possibilities (e.g., t-RNAs) and other oligonucleotides were bound specifically by polypeptides in nature, very little attention had been focused on the non-informational capacities of oligonucleotides. For this reason, among others, little consideration had been given to using oligonucleotides as pharmaceutical compounds.
There are currently at least three areas of exploration that have led to extensive studies regarding the use of oligonucleotides as pharmaceutical compounds. In the most advanced field, antisense oligonucleotides are used to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions. Additionally, the discovery of RNA species with catalytic functions--ribozymes--has led to the study of RNA species that serve to perform intracellular reactions that will achieve desired effects. And lastly, the discovery of the SELEX process Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk and Gold (1990) Science 249:505) has shown that oligonucleotides can be identified that will bind to almost any biologically interesting target.
The use of antisense oligonucleotides as a means for controlling gene expression and the potential for using oligonucleotides as possible pharmaceutical agents has prompted investigations into the introduction of a number of chemical modifications into oligonucleotides to increase their therapeutic activity and stability. Such modifications are designed to increase cell penetration of the oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA and to improve their pharmacokinetic properties. For example, PCT Patent Application Publication No. WO 91/14696, entitled "Oligonucleotide-Transport Agent Disulfide Conjugates," describes a method for chemically modifying antisense oligonucleotides to enhance entry into a cell.
A variety of methods have been used to render oligonucleotides resistant to degradation by exonucleases. PCT Patent Application Publication No. WO 90/15065, entitled "Exo nuclease-Resistant Oligonucleotides and Methods for Preparing the Same," describes a method for making exonuclease-resistant oligonucleotides by incorporating two or more phosphoramidite and phosphoromonothionate and/or phosphorodithionate linkages at the 5' and/or 3' ends of the oligonucleotide. PCT Patent Application Publication No. WO 91/06629, entitled "Oligonucleotide Analogs with Novel Linkages," describes oligonucleotide compounds with one or more phosphodiester linkages between adjacent nucleotides replaced by a formacetal/ketal type linkage which are capable of binding RNA or DNA.
A common strategy for the stabilization of RNA against endonucleolytic cleavage is to modify the 2'-position of ribonucleotides. Interference with base recognition by enzymes can be used to approach stabilization against base-specific endonucleolytic cleavage. Several strategies for this modification are known, including modification with 2'-amino and 2'-fluoro (Hobbs et al. (1973) Biochemistry 12:5138; Guschlbauer et al. (1977) Nucleic Acids Res. 4:1933), and 2'-OCH.sub.3 (Shibahara et al. (1987) 15:4403; Sproat et al. (1989) Nucleic Acids Res. 17:3373). PCT Patent Application Publication No. WO 91/06556, entitled "2' Modified Oligonucleotides," describes nuclease-resistant oligomers with substituents at the 2' position. PCT Patent Application Publication No. WO 91/10671, entitled "Compositions and Methods for Detecting and Modulating RNA Activity and Gene Expression," describes antisense oligonucleotides chemically modified at the 2' position and containing a reactive portion capable of catalyzing, alkylating, or otherwise effecting the cleavage of RNA, a targeting portion, and a tether portion for connecting the targeting and reactive portions.
The 5-position of pyrimidines may also be chemically modified. The introduction of modifications at the C-5 position of pyrimidines may be envisioned to interfere with the recognition by pyrimidine specific endonucleases. However, this concept is not as clear cut as the modification of the 2'-position of ribonucleotides. The first examples of 5-position pyrimidine modifications were demonstrated by Bergstrom (Bergstrom et al. (1976) J. Am. Chem. Soc. 98:1587, (1978) J. Org. Chem. 43:2870, (1981) J. Org. Chem. 46:1432 and 2870, (1982) J. Org. Chem. 47:2174) and Daves (Arai and Daves (1978) J. Am. Chem. Soc. 100:287; Hacksell and Daves (1983) J. Org. Chem. 48:2870). Bergstrom and Daves used 5-mercurial-deoxyuridine compounds, the same as those used by Dreyer and Dervan (1985) Proc. Natl. Acad. Sci. USA 82:968, to tether functional groups to oligonucleotides. A superior method for 5-position modification of pyrimidines is described in U.S. patent application Ser. No. 08/076,735, filed Jun. 14, 1993, entitled "Method for Palladium Catalyzed Carbon-Carbon Coupling and Products," now U.S. Pat. No. 5,428,149 and U.S. patent application Ser. No. 08/458,421, filed Jun. 2, 1995, entitled "Palladium Catalyzed Nucleoside Modifications Using Nucleophiles and Carbon Monoxide," now U.S. Pat. No. 5,719,273, each of which is herein incorporated by reference in its entirety.
A method for simple carbon-carbon coupling reactions to the 5-position of uridines is described in the work of Crisp (1989) Syn. Commun. 19:2117. Crisp forms deoxyuridines functionality at the 5-position by reacting protected 5-iodo-2'-deoxyuridine with alkenylstannanes in acetonitrile in the presence of a Pd (II) catalyst.
To date, very little work has been done to modify purine nucleosides using palladium catalysis. Van Aeroschot et al. (1993) J. Med. Chem 36:2938-2942, report that 2-, 6- and 8-halogenated adenosines can be modified with symmetric organotin reagents. However, symmetric organotin compounds are not widely available. Sessler et al. (1993) J. Am. Chem. 115:10418-10419, describe the arylation of protected 8-bromoguanosine with 4-tributyltinbenzaldehyde. Using this procedure, however, a significant amount of starting material (28%) was unreacted. A superior method for modifying purine nucleosides using palladium catalysts is described in U.S. patent application Ser. No. 08/347,600, filed Dec. 1, 1994, entitled "Purine Nucleoside Modifications by Palladium Catalyzed Methods," now U.S. Pat. No. 5,580,972, and U.S. patent application Ser. No. 08/458,421, filed Jun. 2, 1995, entitled "Palladium Catalyzed Nucleoside Modifications Using Nucleophiles and Carbon Monoxide," now U.S. Pat. No. 5,719,273, each of which is herein incorporated by reference in its entirety.
Additionally, very little work has been done in the area of palladium catalyzed amidations. Schoenberg et al. (1974) J. Org. Chem. 39:3327, describe amidation of aryl and alkenyl halides, however, this work does not include nucleoside substrates or the use of a PdL.sub.4 catalyst.
The palladium-catalyzed coupling of allylic and non-allylic unsaturated alcohols with aryl halides has been explored for a number of years. (See, Kao et al. (1982) J. Org. Chem. 47:1267; Larock et al. (1989) Tetrahedron Lett. 30:6629; Larock (1990) Pure & Appl. Chem. 62:653-660). This reaction provides a method for the preparation of long chain aryl substituted aldehydes and ketones as illustrated below. ##STR1## This reaction has been determined to proceed by the arylpalladation of the alkene, palladium migration and finally palladium hydride elimination to an enol which tautomerizes to the observed carbonyl product. To date, this reaction has not been extended to the functionalization of nucleosides.
SELEX.TM. (Systematic Evolution of Ligands for EXponential Enrichment) is a method for identifying and producing nucleic acid ligands, termed "nucleic acid antibodies" or "aptamers," e.g., nucleic acids that selectively bind to target molecules (Tuerk and Gold (1990) Science 249:505). The method involves selection from a mixture of candidates and step-wise iterations of structural improvement, using the same general selection theme, to achieve virtually any desired criterion of affinity and selectivity. Starting from a mixture of nucleic acids, the method includes steps of contacting the mixture with the target under conditions favorable for interaction, partitioning non-interacting nucleic acids from those nucleic acids which have interacted with the target molecules, dissociating the nucleic acid-target pairs, amplifying the nucleic acids dissociated from the nucleic acid-target pairs to yield a mixture of nucleic acids enriched for those which interact with the target, then reiterating the steps of interacting, partitioning, dissociating and amplifying through as many cycles as desired.
The methods of the present invention may be combined with the SELEX process (See U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096, which is a continuation-in-part of U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, each of which is specifically incorporated herein by reference in its entirety) or the parallel SELEX process (See U.S. patent application Ser. No. 08/309,245, filed Sep. 20, 1994, entitled, "Parallel SELEX," now U.S. Pat. No. 5,723,289; U.S. patent application Ser. No. 08/618,700, filed Mar. 20, 1996, entitled "Parallel SELEX," each of which is specifically incorporated by this reference in its entirety) to produce nucleic acids containing modified nucleotides. The presence of modified nucleotides may result in nucleic acids with an altered structure exhibiting an increased capacity to interact with target molecules. The steric and electronic influence of modified nucleotides may also act to prevent nuclease degradation.